The “angiogenesis” refers to formation of new blood vessels in the body. For angiogenesis, there are a promoter and an inhibitor, and their balance regulates angiogenesis. It is said that the total length of the blood vascular system in an adult reaches 10 km, the surface area of vascular endothelial cells is 7000 m2, and the weight thereof is 1 kg, and the blood vascular system is considered as the largest organ in human, which is distributed in every site in the living body. Angiogenesis occurs vigorously in an embryonic stage or in a process of growth, but in an adult body, angiogenesis is not observed except for special cases such as ovulation and wound healing. The blood vascular system is essential for maintaining the life, and the angiogenesis is a normal reaction in the living body.
On the other hand, abnormal angiogenesis other than that described above causes various diseases. The typical example is cancer angiogenesis. Angiogenesis in cancer tissues brings about a significant increase in cancer as well as hyper-metastasis. Accordingly, if angiogenesis through which nutrients are supplied into cancer cells were inhibited, the cancer could be kept in tumor dormancy. Folkman et al., who proposed tumor dormancy, have identified angiogenesis inhibitors produced by cancer cells and named them angiostatin and endostatin (Non-patent document 1: 0′ Reilly et al., Cell, USA, Vol. 79, No. 2, pp. 315-328, Oct. 21, 1994, Non-patent document 2: 0′ Reilly et al., Cell, USA, Vol 88, No. 2, pp. 277-285, Jan. 24, 1997). It was revealed that these angiogenesis inhibitors almost completely regress cancers in mice, and thereafter angiogenesis has been extensively studied. At present, many pharmaceutical companies are struggling to develop angiogenesis inhibitors and are conducting clinical tests on cancer, but do not arrive at success in overcoming cancer, angiogenesis inhibitors.
Abnormal angiogenesis also occurs in other diseases than cancer, for example diabetic retinopathy and rheumatoid arthritis, and there are a large number of reported data suggesting a possibility that such diseases can be cured by inhibiting angiogenesis. It is therefore estimated that angiogenesis inhibitors can serve as therapeutic agents not only for cancer but also for other diseases accompanied by angiogenesis.
The blood vascular system is distributed in every tissue in the body, but there are also tissues lacking in vascular network. The tissues lacking in vascular network include a cartilage, tendon, ligament and eye ball. In mesenchyme tissues, bone and muscle are rich in blood vessels and have an ability to regenerate them upon bone fracture or damage to muscle.
In the mesenchyme tissues, however, the cartilage, tendon and ligament are tissues lacking in vascular network and are hardly naturally cured upon damage or breakage. These blood-free tubular tissues are destroyed by infiltration with blood vessels, so these tissues are estimated to have an intrinsic angiogenesis inhibitor inhibiting infiltration with surrounding blood vessels.
Chondromodulin-I (ChM-I) is an angiogenesis inhibitor present in cartilage, which is purified being an about 25-kDa glycoprotein purified from fetal bovine cartilage. It is estimated to regulate infiltration of cartilage with blood vessels (Non-patent document 3).
ChM1L, being as a II-type transmembrane protein having homology with ChM-I , is found (Non-patent document 4: Yamana et al., Biochemical and Biophysical Research Communications, USA, Vol. 280. No. 4, pp. 1101-1106, Feb. 2, 2001, Patent document 1: WO 01/23557) connective tissues such as tendon and ligament and is estimated to regulate infiltration of these tissues with blood vessels (Patent document 2: WO 01/53344, Non-patent document 5: Brandau et al., Developmental Dynamics: an official publication of the American Association of Anatomists , USA, Vol. 221, No. 1, pp. 72-80, May, 2001, Non-patent document 6: Shukunami et al., Biochemical and Biophysical Research Communications, USA, Vol 280, No. 5, pp. 1323-1327, Feb. 2, 2001).
As described above, the tendon and ligament as well as the cartilage are tissues lacking in vascular network. The tendon and ligament are very important tissues connecting bones and muscles, and their damage or breakage is considered as a serious disease by which not only athletes but also ordinary people are subject to limitation in physical exercise. Thus, tendon and ligament tissues are important tissues, but as compared with cartilage, are not so investigated in fundamental and clinical studies. This is due to the absence of marker molecules expressed specifically in the tendon or ligament, in addition to difficult acquisition of tendon and ligament materials such as cells. Under these circumstances, there is demand for marker molecules capable of evaluating the degree of damage or repair in the tendon and ligament. ChM1L is expressed specifically in the tendon and ligament and is thus considered usable as a marker molecule for evaluating damage or repair in these tissues. There is also a possibility that by regulating the activity of ChM1L, damage to the tendon or ligament can be treated.
For use of ChM-I, its secretory protein and a protein such as ChM1L as angiogenesis inhibitors, however, there are many problems to be solved.
For development of a recombinant protein as a pharmaceutical preparation, its active protein should be prepared in a large amount. Generally, an expression system of using a microorganism, particularly Escherichia coli, is widely used in protein production in industrial scale. Expression in Escherichia coli is advantageous in that a very high level of recombinant protein can be obtained by using a vector capable of high-level expression as well as culture in high density.
However, Escherichia coli often produce an inclusion body containing the recombinant protein, which is a serious problem when Escherichia coli is used as a host. Actually, Escherichia coli produce an inclusion body therein of endostatin examined as an angiogenesis inhibitor in a clinical test, and its refolding is difficult (Non-patent document 2: 0′ Reilly et al., Cell, USA, Vol 88, No. 2, pp. 277-285, Jan. 24, 1997), so a modified method is still researched at present (Patent document 3: Japanese Patent Application National Publication (Laid-Open) No. 2002-504494).
Development of ChM-I as anticancer drug by utilizing its inhibitory activity on angiogenesis has also been examined, but it is reported that Escherichia coli produce an inclusion body of ChM-I upon expression therein which is hardly refolded (Non-patent document 7: Yamakawa et al., The Molecular Biology Society of Japan, 25 th annual meeting, Collection of Lecture Abstracts, 2P-0206, Nov., 2001). Even if Chinese hamster ovary (CHO) cells are used as host cells, ChM-I forms an aggregation and thus requires a refolding process, which makes acquisition of a large amount of its active protein difficult (Non-patent document 8: Azizan et al., The Journal of Biological Chemistry, USA, Vol. 276, No. 26, pp. 32419-32426 Jun. 29, 2001).
It is known that Escherichia coli produce an inclusion body of ChM1L, as in the case of ChM-I , upon expression therein which is hardly refolded (Non-patent document 9: Hasegawa et al., The Molecular Biology Society of Japan, 25 th annual meeting, Collection of Lecture Abstracts, 2P-0770, Nov., 2001) . ChM1L can be obtained as an active protein from culture medium when it is expressed in COS7 cells. Its expression level is, however, low and an aggregation of ChM1L is observed as in the case of ChM-I, which makes acquisition of a large amount of its active protein impossible (Patent document 4: WO 00/12708) .
In some cases, protein is cleaved and secreted extracellularly. For example, it is known that tumor necrosis factor-α (TNF-α) is expressed an extracellular domain of a transmembrane as a type-II transmembrane protein and functions as a transmembrane protein, but is cleaved with a protease such as TNF-α converting enzyme and also functions as a secretory protein. It is also known that ChM-I has a type-II membrane protein structure, but undergoes processing at a protease (e.g. furin) recognition site (REHR) to secrete its C-terminal 120 amino acids extracellularly (Non-patent document 3: Hiraki et al. , The Journal of Biological Chemistry, USA, Vol. 272, No. 51, pp. 32419-32426, Dec. 19, 1997, Non-patent document 8: Azizan et al., The Journal of Biological Chemistry, USA, Vol. 276, No. 26, pp. 32419-32426 Jun. 29, 2001)
However, the secretory ChM-I consisting of the above 120 amino acid residues, even when produced by recombination, does not exhibit sufficient solubility and fails to solve the problem described above.
ChM1L has homology in amino acid sequence with ChM-I, but is free of a typical protease recognition site like what ChM-I has and is estimated to function as a transmembrane protein, and there is no report where ChM1L is found to be a secretory protein (Patent document 4: WO 00/12708, Patent document 5: WO 00/29579, Patent document 6: WO 01/23557, Patent document 7: WO 01/48203, Patent document 8: WO 01/53344, Non-patent document 4: Yamana et al., Biochemical and Biophysical Research Communications, USA, Vol. 280. No. 4, pp. 1101-1106, Feb. 2, 2001, Non-patent document 5: Brandau et al., Developmental Dynamics: an official publication of the American Association of Anatomists , USA, Vol. 221, No. 1, pp. 72-80, May, 2001, Non-patent document 6: Shukunami et al. , Biochemical and Biophysical Research Communications, USA, Vol280, No. 5, pp. 1323-1327, Feb. 2, 2001).    Patent document 1: WO 01/23557    Patent document 2: WO 01/53344    Patent document 3: Japanese Patent Application National Publication (Laid-Open) No. 2002-504494    Patent document 4: WO 00/12708    Patent document 5: WO 00/29579    Patent document 6: WO 01/23557    Patent document 7: WO 01/48203    Patent document 8: WO 01/53344    Non-patent document 1: O' Reilly et al., Cell, USA, Vol. 79, No. 2, pp. 315-328, Oct. 21, 1994    Non-patent document 2: O' Reilly et al., Cell, USA, Vol. 88, No. 2, pp. 277-285, Jan. 24, 1997    Non-patent document 3: Hiraki et al., The Journal of Biological Chemistry, USA, Vol. 272, No. 51, pp. 32419-32426, Dec. 19, 1997    Non-patent document 4: Yamana et al., Biochemical and Biophysical Research Communications, USA, Vol. 280. No. 4, pp. 1101-1106, Feb. 2, 2001    Non-patent document 5: Brandau et al., Developmental Dynamics: an official publication of the American Association of Anatomists, USA, Vol. 221, No. 1, pp. 72-80, May, 2001    Non-patent document 6: Shukunami et al., Biochemical and Biophysical Research Communications, USA, Vol. 280, No. 5, pp. 1323-1327, Feb. 2, 2001    Non-patent document 7: Yamakawa et al., The Molecular Biology Society of Japan, 25th annual meeting, Collection of Lecture Abstracts, 2P-0206, November, 2001    Non-patent document 8: Azizan et al., The Journal of Biological Chemistry, USA, Vol. 276, No. 26, pp. 32419-32426, Jun. 29, 2001    Non-patent document 9: Hasegawa et al., The Molecular Biology Society of Japan, 25th annual meeting, Collection of Lecture Abstracts, 2P-0770, November, 2001